Helica Elisa Kit

1. FUMONISIN ELISA ASSAY
FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
(Cat. No. 951FUM01C-96)
FUMONISINS
The fumonisins (B1, B2, and B3) are a group of mycotoxins produced by Fusarium moniliforme.
Fumonisins have been found world-wide as a contaminant of maize and have been shown to
cause liver cancer in experimental rats, pulmonary edema in pigs and leukoencephalomalacia in
horses. High levels of fumonisins in locally grown maize have been found in areas of the world
which have a high prevalence of human esophageal cancer, for instance, in South Africa and
China.
INTENDED USE
The HELICA BIOSYSTEMS fumonisin ELISA is a competitive enzyme-linked immunoassay
intended for the quantitative detection of fumonisins in maize. For research use only. Not for use
in diagnostic procedures.
ASSAY PRINCIPLE
The HELICA BIOSYSTEMS fumonisin ELISA is a solid phase direct competitive enzyme
immunoassay. A fumonisin-specific antibody optimized to cross react with the three fumonisin
subtypes is coated to a polystyrene microwell. Toxins are extracted from a ground sample with
90% methanol. The extracted sample and HRP-conjugated fumonisin are mixed and added to the
antibody-coated microwell. Fumonisin from the extracted sample and HRP-conjugated fumonisin
compete to bind with the antibody coated to the microwell. Microwell contents are decanted and
non-specific reactants are removed by washing. An enzyme substrate (TMB) is added and color
(blue) develops. The intensity of the color is directly proportional to the amount of bound
conjugate and inversely proportional to the concentration of fumonisin in the sample or standard.
Therefore, as the concentration of fumonisin in the sample or standard increases, the intensity of
the blue color will decrease. An acidic stop solution is added which changes the chromagen color
from blue to yellow. The microwells are measured optically by a microplate reader with an
absorbance filter of 450nm (OD450). The optical densities of the samples are compared to the
OD’s of the kit standards and an interpretative result is determined.

2. 2
REAGENTS PROVIDED
MATERIALS REQUIRED BUT NOT PROVIDED
Extraction Procedure
 Grinder sufficient to render sample to
particle size of fine instant coffee
 Collection Container: Minimum 125mL
capacity
 Balance: 20g measuring capability
 Graduated cylinder: 100mL
 Methanol: 36mL reagent grade per
sample
 Distilled or deionized water: 4mL per
sample
 Filter Paper: Whatman #1 or equivalent
 Filter Funnel
Assay Procedure
 Pipettor with tips: 100µL and 200µL
 Timer
 Wash bottle
 Absorbent paper towels
 Microplate reader with 450nm filter
PRECAUTIONS
1. Bring all reagents to room temperature
(19º - 27ºC) before use.
2. Store reagents at 2 to 8C, and do not
use beyond expiration date(s). Never
freeze kit components.
3. Do not return unused reagents back into
their original bottles. The assay
procedure details volumes required.
4. Adhere to all time and temperature
conditions stated in the procedure.
5. Never pipette reagents or samples by
mouth.
6. The Stop Solution contains acid. Do not
allow to contact skin or eyes. If exposed,
flush with water.
7. Consider all materials, containers and
devices that are exposed to sample or
standards to be contaminated with
fumonisin. Wear protective gloves and
safety glasses when using this kit.
8. Dispose of all materials, containers and
devices in the appropriate receptacle
after use.
9. HRP-labeled conjugate and TMB-
substrate are photosensitive and are
packaged in a protective opaque bottle.
Store in the dark and return to storage
after use.
1 X Pouch
Antibody Coated
Microwell Plate
96 wells (12 eight well strips) in a microwell holder coated with
a mouse anti-fumonisin monoclonal antibody, Ready-to-Use.
1 X Plate Mixing Wells Green
96 non-coated wells (12 eight well strips) in a microwell holder,
Ready-to-Use.
6 X Vial
Fumonisin
Standards
Black Cap
1.5mL/vial of fumonisin at the following concentrations: 0.0,
2.5, 7.5, 20.0, 50.0, 150.0 ng/mL in aqueous solution, Ready-
to-Use.
1 X Bottle
Streptavidin HRP
Conjugate A
Green Cap
12mL of peroxidase conjugated streptavidin in buffer with
preservative, Ready-to-Use.
1 X Bottle
Biotinylated
Fumonisin
Conjugate B
White Cap
12mL of biotinylated fumonisin in buffer with preservative,
Ready-to-Use.
1 X Bottle
Substrate
Reagent
Blue Cap 12mL stabilized tetramethylbenzidine (TMB), Ready-to-Use.
1 X Bottle Stop Solution Red Cap 12mL Acidic Solution, Ready-to-Use.
1 X Pouch Washing Buffer
PBS with 0.05% Tween20, bring to 1 liter with distilled water
and store refrigerated.

3. 3
KIT SAFETY AND WASTE DISPOSAL INSTRUCTIONS
Mycotoxins (aflatoxins, trichothecenes, and others) are well known carcinogens in humans and
are thus considered highly toxic. Do not dispose of these materials down the drain.
Please note that there is a potential for mycotoxin contamination in or on any of the kit
components provided. It is strongly advised that gloves, a lab coat, and safety glasses be worn at
all times while handling mycotoxin kits and their respective components.
1. Before conducting the assay, prepare a waste container to act as a receptacle for your kit
waste. Eject contaminated pipette tips and all other related materials into this container.
2. Once the assay is completed, the container should be treated with a sufficient amount of 5-
6% sodium hypochlorite (NaOCl) to saturate the contents of the container (approximately
1/10th the volume of the container). 5-6% NaOCl will denature the mycotoxins and neutralize
the waste, making it safe for disposal. Invert the container several times to thoroughly coat all
waste.
3. In the case of an accidental toxin spill, treat the spill surface with 5-6% NaOCl for a minimum
of 10 minutes followed by 5% aqueous acetone. Wipe dry with absorbent paper towels.
EXTRACTION PROCEDURE
Note: The sample must be collected according to established sampling techniques
1. Prepare the Extraction Solution (90%
Methanol) by adding 4mL of distilled or
deionized water to 36mL of methanol
(reagent grade) for each sample to be
tested.
2. Grind a representative sample to the
particle size of fine instant coffee (95%
passes through a 20 mesh screen).
3. Weigh out a 20g ground portion of the
sample and add 40mL of the Extraction
Solvent (90% methanol).
Note: The ratio of sample to extraction
solvent is 1:2 (w/v).
4. Mix by shaking in a sealed container or
in a blender for one minute.
5. Allow the particulate matter to settle,
then filter 5 - 10mL of the extract
through a Whatman #1 filter paper (or
equivalent) and collect the filtrate to be
tested.
6. Dilute the sample extract 1:20 in distilled
water (e.g. 0.1mL + 1.9mL)
7. Diluted sample is now ready for testing.
ASSAY PROCEDURE
Note: It is recommended that a multi-channel pipettor be utilized to perform the assay. If a single
channel pipettor is used, it is recommended that no more than a total of 16 samples and
standards (2 test strips) are run.
1. Bring all the reagents to room temperature before use. Reconstitute the PBS-Tween packet
by washing out the contents with a gentle stream of distilled water into a 1-Liter container.
2. Place one Dilution Well in a microwell holder for each Standard and Sample to be tested.
Place an equal number of Antibody Coated Microtiter Wells in another microwell holder.
3. Dispense 100L of the Conjugate solution A (green) into the appropriate dilution wells
followed by 100L of Conjugate solution B (clear).
4. Using a new pipette tip for each, add 100L of each Standard and Sample to appropriate
Dilution Well containing Conjugate mixture. Mix by priming pipettor 3 times.
Note: Operator must record the location of each Standard and Sample throughout test.
5. Using a new pipette tip for each, transfer 100L of contents from each Dilution Well to a
corresponding Antibody Coated Microtiter Well. Incubate at room temperature for 10 minutes.
The mixing wells contain enough solution to run each standard and/or sample in duplicate if
so desired.
6. Decant the contents from microwells into a discard basin. Wash the microwells by filling each
with PBS Tween wash buffer then decanting the buffer into a discard basin. Repeat wash for
a total of 5 washes.
7. Tap the microwells (face down) on a layer of absorbent towels to remove residual buffer.
8. Measure the required volume of Substrate Reagent (1mL/strip or 120L/well) and place in a
separate container. Add 100L to each microwell. Incubate at room temperature for 10
minutes. Cover to avoid direct light.

4. 4
9. Measure the required volume of Stop Solution (1mL/strip or 120L/well) and place in a
separate container. Add 100L in the same sequence and at the same pace as the Substrate
was added.
10. Read the optical density (OD) of each microwell with a microtiter plate reader using a 450nm
filter. Record the optical density (OD) of each microwell.
INTERPRETATION OF RESULTS
Construct a dose-response curve using either the unmodified OD values or the OD values
expressed as a percentage of the OD of the zero (0.0) standard against the fumonisin content of
the standard. Unknowns are measured by interpolation from the standard curve.
The information contained on the label of a standard vial refers to the contents of that vial.
However, the sample has been diluted at a 2:1 ratio with 90% methanol followed by a dilution of
20:1 in distilled water, and so the level of fumonisin shown by the standard must be multiplied by
40 in order to indicate the ng of fumonisin per gram of commodity (ppm) as follows:
standard ng/mL Commodity µg/gm (ppm)
0.0 0.0
2.5 0.1
7.5 0.3
20.0 0.8
50.0 2.0
150.0 6.0
The sample dilution results in a standard curve from 0.0 ppm to 6.0 ppm. If a sample contains
fumonisin at greater than the highest standard, it should be diluted appropriately in distilled water
and retested. The extra dilution step should be taken into consideration when expressing the final
result.
SAMPLE HPLC HELICA ELISA
1 <0.1 ppm <0.1 ppm
2 0.6 +/- 0.2 ppm 0.72 +/- 0.06 ppm
3 2.0 +/- 0.3 ppm 1.98 +/- 0.12 ppm
4 3.5 +/- 0.4 ppm 3.30 +/- 0.35 ppm
___________________
HELICA BIOSYSTEMS, INC.
v.04D July 2019